Molecular imaging (MI) enables integration of patient and disease-related information with anatomical imaging (Hanson, R. N., 2012). With MI one can visualize, characterize, and measure biological processes at the molecular level in a living organism (Kodibagkar, V. D et al., 2012; Koolen, B. B. et al., 2012; Misri, R. et al., 2012). Further, use of MI in the diagnosis of a disease results in improved clinical risk assessment, optimization of treatment and therapy, better patient outcome, and helps inter- and intra-subject comparisons. (Moss, J. A. et al., 2012; Vavere, A. L. et al., 2012; James, M. L. et al., 2012).
In medicine MI has evolved into two distinctive areas: anatomy-based or structural imaging, and nuclear medicine (Beyer, T. et al., 2009; Antoch, G., et al., 2009; Mawlawi, O. et al., 2009). Traditional imaging such as computed tomography (CT) and magnetic resonance imaging (MRI) are now primarily used for extracting anatomical and/or structural information. On the other hand nuclear medicine, using methods such as positron emission tomography (PET) and single photon emitted tomography (SPECT)), focusses on molecular events in living organisms, and thereby provides functional and/or physiological imaging. In oncology the information derived from PET and SPECT imaging is integrated with other patient specific disease information for diagnosis and treatment (Buck, A. K. et al., 2012; Morales-Avila, E. et al., 2011; Khalil, M. M. et al., 2011). Table 1 below summarizes the major differences among the various MI modalities.
TABLE 1Noninvasive in vivo molecular imaging modalities (Buck, A. K. et al., 2012)Sensitivity ModalitySpatial AcquisitionProbe ofDepth ofEnergy Resolution (mm)time/frame massdetectionpenetrationImagingusedClinicalanimal(s)(ng)(Mol/l))(mm)PETAnnihilation3-81-3 1-300 1-10010−11-10−12>300SPECTΥ-photons 5-121-4 60-2000  1-100010−10-10−11>300CTX-rays0.5-1  0.03-0.4  1-300——>300MRIRf0.2-0.10.025-0.1  50-3000103-10610−3-10−5>300UltrasoundHigh Rf0.1-1.00.05-0.1  0.1-100 103-106— 1-200BLIIR— 3-10 10-300103-10610−13-10−161-10FLIIR— 3-10 10-2000103-106 10−9-10−111-20
Magnetic resonance imaging (MRI) primarily provides information about soft tissue. Although MRI provides high spatial resolution (<1 mm), and has the ability to visualize anatomical, physiological, and/or metabolic information in a single imaging session, it has low sensitivity for biological targets. Therefore, this method has not been tailored to visualize cancer or disease specific disorders (Misri, R. et al., 2012).
X-ray computed tomography or computed tomography (CT scan) is a computer processed X-ray imaging technology that provides anatomical images of the body, which may be manipulated to obtain diagnostic information. CT inherently provides high-contrast and resolution, but exposes the patient to high radiation dose enhancing the risk of DNA or cellular damage. CT methods that provide high signal to noise ratio at lower radiation doses are currently being investigated (Buck, A. K. et al., 2012).
Optical imaging is another modality for molecular imaging, and is based on the detection of light photons and their interaction with the tissue. It has been used mostly in preclinical studies. Two major optical imaging methods are bioluminescence imaging (BLI) and fluorescence imaging (FI). BLI requires cellular expression of the enzyme luciferase, which is incorporated into the DNA of animals used for modeling of any given disease. BLI is most useful in vivo in small animal disease models, e.g., mouse models, since the depth sensitivity of the BLI is only 1-2 cm (Alhasan, M. K., et al. 2012). Fluorescent imaging (FI) is designed to look at the cell surface distribution of fluorescent signals. It is used in both live and fixed cells, and does not require any substrate. In this method fluorochromes are conjugated to peptides or antibodies, and binding of the conjugates to targets on the surface of cells provides information about target expression. Fluorescent imaging provides contrast sensitivity that is approximately comparable to radioactivity-based imaging. However, tissue penetration of FI signals is less than that of PET or SPECT. New methods of FI for improving relative sensitivity and resolution, preparation of newer imaging probes, and better signal-to-noise amplifiers are currently being developed (Kodibagkar, V. D. et al., 2012; Koolen, B. B. et al., 2012; Misri, R. et al., 2012).
Ultrasound imaging (UI) also a molecular imaging technique, and is currently being development as a nano-drug delivery system. UI is performed by utilizing microbubbles, liposomes, or perfluorocarbon emulsions as scaffolds functionalized with different targeting agents. UI offers high spatial resolution (<1 mm) and can provide detailed anatomical information for coregistration with other molecular imaging methods. Because of the relatively large size of imaging reagents or particles (>250 nm) this technique has limited capacity for tissue penetration, and is used specifically for vascular imaging.
Molecular imaging of estrogen receptor expressing cells and tissues can provide the ability to detect tumors that overexpress the estrogen receptor (ER), having significant positive impact on diagnosis and therapy of ER-dependent cancer, such as breast cancer, in which over-expression of ER plays a major role (Dunn L. et al., 2009; Varghese C. et al., 2007; Schiff, R. et al., 2005). Among patients presenting with breast cancer, approximately 65-75% have primary tumors with elevated ER levels. Currently, receptor status of the tumor is determined via fine needle biopsy and, while minimally invasive, the technique requires removing tumor cells to be analyzed from the body (Higa, G. M. et al., 2009; Allred, D. C. et al., 2009). Because treatment protocols are dependent upon accurate characterization of the tissue sample, alternate methods that non-invasively characterize the entire body for potential distal metastases, and are quantitative, are of interest. MI agents that bind to specific biomarkers can provide important physiological and biochemical information regarding the disease leading to improved treatment plans and improved patient outcomes.
Several non-invasive modalities are currently used to image normal and cancerous breast tissue (Keune, J. D. et al., 2010; DeMartini, W. et al., 2008; van de Ven, S. M. W. Y. et al., 2008; Yu, E. Y. et al., 2007; Czernin, J. et al., 2010; Pantaleo, M. A. et al., 2008). Although many biomarkers associated with tumor sensitivity and invasiveness are being examined using experimental radiotracers, the expression of ER remains one of the most important diagnostic indicators for breast cancer (de Vries, E. F. J. et al., 2007; Schroeder, C. P. et al., 2007; Dittmann, H. et al., 2009). A radiotracer that binds with high affinity and selectivity to the estrogen receptor-alpha (ERα) would yield diagnostic data not provided by any other molecular agent or imaging modality as it would directly indicate the receptor status of suspected lesions. Whole body imaging would detect other ER-expressing tissues which may be secondary lesions. Imaging following chemotherapy (and/or surgery) would also demonstrate the presence or absence of recurrent ER-responsive disease, as well as the effectiveness of the intervention. Such information is crucial in providing individualized treatment.
Estradiol derivatives used for molecular imaging in clinical studies include radioiodinated molecules, e.g., isomers of 11β-methoxy-(17α,20E/Z)-[123I]iodovinylestradiol. Among the isomers, the 20Z isomer yields better images of ER(+) human breast tumors than the 20E isomer. Although the diagnostic radioiodinated estradiols were selectively and sensitively detected in both primary and metastatic ER(+) breast cancer, extensive correlation between imaging and clinical outcome has not yet become available (Pysz, M. A. et al., 2010; Yager, J. D. et al., 2006; Benard, F. et al., 2005; Cummins, C. H., 1993).
Steroidal estrogen derivatives with the prosthetic 99mTc chelates at different positions have been investigated also, although none have yet been clinically approved (Pavlik, E. J. et al., 1990).
Estradiol derivatives labeled with 18F have also been explored, and among the compounds evaluated (FIG. 1), 16α-[18F]-estradiol ([18F]-ES) is currently in clinical use. The 11β analog, 4-Fluoro-11β-methoxy-16α-[18F]-flouroestradiol (4FMFES; FIG. 1) tracer also showed favorable biodistribution in small animal studies, and exhibited higher selectivity uptake ratios than that obtained by using [18F]-ES. The compound 4FMFES is also being evaluated in studies for human biodistribution and dosimetry. Another compound, 7α-substituted [18F]-ES, has also been prepared and evaluated as a possible tracer for ER(+) breast tumors. However, the biological data showed no significant improvement on target to non-target uptake ratios compared to other 18F-ES radiotracers (Cummins, C. H., 1993; Pavlik, E. J. et al., 1990; Ribeiro-Barras, M. J. et al., 1992; Rijks, L. J. M. et al., 1997; Bennink, R. J. et al., 2001).
Although [18F]-16α-fluoroestradiol (FES) was developed in the 1980s, its use has been limited to fewer than a thousand patients (Kiesewetter, D. O. et al., 1984; Kiesewetter, D. O., Kilbourn, M. R. et al., 1984; Sundararajan, L. et al., 2007; Kurland, B. F. et al., 2011; Peterson, L. M. et al., 2011; Linden, H. M. et al., 2011; Dehdashti, F. et al., 2009; Gemignani, M. L., et al., 2013). FES is prepared from a simple precursor (2 mg/run) in an overall radiochemical yield of only approximately 30%. A careful chromatographic separation is required to remove several other radioactive by-products and non-radioactive materials. FES is typically obtained with a specific activity in the 200-1000 Ci/mmol range, appropriate for most clinical applications. This radiosynthetic procedure is not trivial, even with the use of automated methods (Lim, J. L. et al., 1996; Romer, J. et al., 1999; Oh, S. J. et al., 2007; Kumar, P. et al., 2007; Kumar, P. et al., 2012; Knott, K. E. et al., 2011). Biologically, FES is essentially a mimic for endogenous estradiol and undergoes comparable pharmacodynamic (PD) and pharmacokinetic (PK) processes (Mankoff, D A. et al., 1997; Jonson, S. D. et al., 1998; Bonasera, T. A. et al., 1997; Downer, J. B. et al., 2001; Benard, F. et al., 2001).
FES binds to steroid hormone binding globulin (SHBG) and as much as 45% of FES in circulating plasma is found complexed with SHBG. Therefore, although FES binds to ER in target tissues with a relative binding affinity (RBA) of 80%, it may largely not be accessible to target tissue, thereby impairing uptake of FES uptake in tumors. FES is rapidly cleared by the liver where it undergoes rapid and extensive metabolism such that less than 20% of radioactivity in plasma of patients is parent FES. The metabolites generated contribute to non-specific tissue distribution and accumulation in the liver and gut, and results in high background signal that compromises detection of lesions in adjacent tissues. Nevertheless, eleven completed or ongoing clinical studies are attempting to demonstrate the extent to which FES can contribute to the management of patients with breast cancer (see clinicaltrials.gov). A recent report shows that imaging with FES of patients with primary breast cancer in a preoperative setting can provide valuable information, but significant limitations still remain (Gemignani, M. L. et al., 2013). For example, the Gemignani, M. L. et al., report showed that although the standard uptake value of 18F-FES PET correlated with ER immunohistochemistry expression, it did not correlate with gene expression patients with early breast cancer.
Imaging of estrogen receptor (ER) in vivo using ER binding radiopharmaceuticals can be used for determining the ER expression status during tumor staging. Fluorine-18, Iodine-123 and other cyclotron-produced radionuclides have been used to label ER binding ligands to develop such in vivo radioimaging probes (Pomper, M. G. et L., 1990; Kiesewetter, D. O. et al., 1984; VanBrocklin, H. F. et al., 1993; VanBrocklin, H. F. et al., 1994; Landvatter, S. W. et al., 1983; French, A. N. et al., 1993; French, A. N., Wilson, S. R. et al., 1993; LaFrate, A. L. et al., 2009; Bergmann, K. E. et al., 1994; Hostetler, E. D. et al., 1999).
Some of these radioligands, for example 16α-18F-17β-estradiol (18F-FES), have been evaluated clinically for the imaging of hormone dependent breast tumor and predicting the responsiveness of the tumor to antiestrogen drugs (Seimbille, Y. et al., 2002; Mortimer, J. E. et al., 1996). So far, none of the established ligands has been approved for routine clinical use as a breast cancer diagnostic reagent. Estrogen imaging agents with the radionuclide 99mTc for SPECT have also been reported (Bigott, H. M. et al., 2005; Luyt, L. G. et al., 2003; Skaddan, M. B.; Wust, F. R.; Jonson, S. et al., 2000). However, most of the reported compounds have displayed suboptimal target tissue selectivity, possibly due to their lipophilicity, or rapid metabolism. In addition, these radioligands were synthesized by inefficient linear approaches, leading to products that were difficult to purify, leading to suboptimal yield and purity (Huang, L. et al., 2010; Nayak, T. K. et al., 2008).
Because many of the radioligands that have been developed still exhibit low receptor (estrogen receptors ERα, ERβ) binding affinity and non-ER regulated uptake, there is a need to develop ER-targeted radioligands having high specific activity (radioactivity per unit mass of the radioligand >1 Ci/mmol, high specific receptor binding affinity, low non-specific binding), and appropriate metabolic and clearance characteristics, both to characterize the tumors and to predict or determine their response to anti-hormonal therapy.